Current clinical criteria for Lynch syndrome are not sensitive enough to identify MSH6 mutation carriers

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Current clinical criteria for Lynch syndrome are not sensitive enough to identify MSH6 mutation carriers

Wenche Sjursen,

^1,2

Bjørn Ivar Haukanes,

³

Eli Marie Grindedal,

⁴

Harald Aarset,

¹

Astrid Stormorken,

⁵

Lars F Engebretsen,

³

Christoffer Jonsrud,

⁶

Inga Bjørnevoll,

¹

Per Arne Andresen,

⁷

Sarah Ariansen,

⁷

Liss Anne S Lavik,

¹

Bodil Gilde,

¹

Inger Marie Bowitz-Lothe,

⁵

Lovise Mæhle,

⁴

Pa ˚l Møller

⁴

ABSTRACT

BackgroundReported prevalence, penetrance and expression of deleterious mutations in the mismatch repair (MMR) genes,MLH1,MSH2,MSH6andPMS2, may reflect differences in the clinical criteria used to select families for DNA testing. The authors have previously reported that clinical criteria are not sensitive enough to identify MMR mutation carriers among incident colorectal cancer cases.

ObjectiveTo describe the sensitivity of the criteria when applied to families with a demonstrated MMR mutation.

MethodsFamilies with an aggregation of colorectal cancers were examined for deleterious MMR mutations according to the Mallorca guidelines. All families with a detected MMR mutation as of November 2009 were reclassified according to the Amsterdam and Bethesda criteria.

ResultsSixty-nine different DNA variants were identified in a total of 129 families. The original Amsterdam clinical criteria were met by 38%, 12%, 78% and 25% of families with mutations inMSH2,MSH6,MLH1andPMS2, respectively. Corresponding numbers for the revised Amsterdam criteria were 62%, 48%, 87% and 38%.

Similarly, each of the four clinical Bethesda criteria had low sensitivity for identifyingMSH6orPMS2mutations.

ConclusionAmsterdam criteria and each of the Bethesda criteria were inadequate for identifyingMSH6 mutation-carrying kindreds.MSH6mutations may be more common than currently assumed, and the penetrance/expression ofMSH6mutations, as derived from families meeting current clinical criteria, may be misleading. To increase detection rate of MMR mutation carriers, all cancers in the Lynch syndrome tumour spectrum should be subjected to immunohistochemical analysis and/or analysis for microsatellite instability.

INTRODUCTION

The concept of hereditary non-polyposis colorectal cancer (HNPCC) was developed to denote families with inherited colorectal cancer (CRC). The Amsterdam (AMSI) criteria identiﬁed families with CRC.¹As extracolonic cancers, especially endometrial cancer, were shown to be part of the inherited syndrome,² ³ the revised Amsterdam criteria (AMSII) were introduced.⁴The Bethesda guidelines included the tumour marker microsatellite instability (MSI),⁵and the revised Bethesda criteria (BII) speciﬁed all cancers known at the time to be asso-

ciated with the syndrome.⁶ Prostate cancer has recently been shown to possibly be part of the syndrome.⁷ Germline mutations in the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2, have been identified to cause HNPCC (reviewed by Lynch and Lynch⁸). However, it has become clear that not all families fulfilling the clinical criteria have an identifiable deleterious mutation (hereafter called ‘mutation’) in one of these genes. In addition, because MMR mutations confer an increased risk of several types of cancer in addition to CRC, it has been suggested that the term Lynch syndrome should replace HNPCC in families where a mutation has been detected.⁹This definition of Lynch syndrome will be used in the present report. Families fulfilling the AMSII criteria without a demonstrable MMR mutation may be denoted HNPCC. Families with an aggregation of CRC and not corresponding to Lynch syndrome or HNPCC may be referred to as familial CRC.⁹

Tumours caused by mutations inMLH1, MSH2, MSH6andPMS2show a high degree of MSI. It has been shown by immunohistochemical analysis (IHC) that the gene product from the mutated gene is absent in tumour tissue (reviewed by Vasenet al¹⁰).

IHC and MSI analysis have high sensitivity in detecting carriers of MMR mutations.^{11 12}It is now customary to examine tumours in families that fulﬁl clinical criteria by IHC/MSI analysis, and select those families with abnormal results for analysis of constitutional DNA.¹⁰ As a consequence, families not meeting the clinical criteria will not be subjected to mutation analyses.

Varying prevalence of mutations in the MMR genes has been reported. Some variations are obvi- ously caused by geographically local and frequent founder mutations.^13e16It is, however, reasonable to assume that the criteria used to select families for testing may also have inﬂuenced the results.

Over the last two decades, Norwegian cancer genetic clinics have recruited families with an aggregation of cancers of any type. Thousands of cancer kindreds have been examined for hereditary cancer syndromes. Reports from this are listed on http://

www.inherited-cancer.com. Upon referral, the families were classiﬁed using preset wide-ranging criteria, and IHC/MSI analyses were performed not only on the families that met the clinical criteria for HNPCC and familial CRC. We here report the sensitivities of the AMSI, AMSII and BII criteria when applied to families that were shown by genetic testing to have an MMR mutation. As Norwegian legislation

1Department of Pathology and Medical genetics, St Olavs University Hospital, Trondheim, Norway

2Department of Laboratory Medicine Children’s and Women’s Health, Norwegian University of Science and Technology, Trondheim, Norway

3Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway

4Department of medical genetics, Oslo University Hospital, Radiumhospitalet, Oslo, Norway

5Department of Medical Genetics, Oslo University Hospital, Ulleva˚l, Norway

6University Hospital of North Norway, Division of Child and Adolescent Health, Department of Medical Genetics, Tromsø, Norway

7Department of Pathology, Oslo University Hospital,

Rikshospitalet, Oslo, Norway Correspondence to Dr Wenche Sjursen, Department of Pathology and Medical Genetics, Erling Skjalgssons gt 1, St Olavs University Hospital, 7006 Trondheim, Norway;

[email protected] Received 29 January 2010 Accepted 27 March 2010 Published Online First 28 June 2010

This paper is freely available online under the BMJ Journals unlocked scheme, see http://

jmg.bmj.com/site/about/

unlocked.xhtml

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dictates that genetic testing is restricted to clinical departments, and as all genetic departments collaborated in this report, we here present a complete report of all clinical genetic activity in a deﬁned population up to November 2009.

PATIENTS AND METHODS Patients, registries and ethics

The initial material included all families investigated for inherited CRC in Norway until November 2009. Wide-ranging selection criteria were used to identify the families. Besides including all kindreds meeting the AMSII or BII criteria, we explored all families with four CRC cases irrespective of age and including skipped generations or with an aggregation of any cancer associated with Lynch syndrome. All activities were conducted as part of the healthcare system, all information was included in the patient ﬁles, all genetic testing was conducted according to national legislation, including genetic counselling before and after genetic testing, and all genetic testing was performed with written informed consent from the participants.

All relevant diagnoses in the families were validated in the medical ﬁles or cancer registry after consent from relatives or descendants if the subject was dead. No research registry that included names was produced; only summarised data were taken from medical ﬁles for compilation of the present report. All information described has been disclosed to the patients/families, and family members were offered appropriate healthcare according to the Mallorca guidelines.⁹

MSI/IHC examinations

Upon referral and inclusion according to the wide-ranging criteria, families were subjected to examination for Lynch syndrome as described in the Mallorca guidelines, with IHC/

MSI analysis of at least two affected family members if available,⁹continuing to full mutation analysis of the relevant gene(s) of the patient (or obligate carrier in the family or offspring if dead) if an abnormal IHC result was obtained. A family was scored as having an abnormal IHC result if one or more tumour(s) showed lack of staining for the gene product of one or more of the MMR genes. Full mutation analysis of all MMR genes was performed if IHC was normal but the tumours were MSI (MSI-high). In some selected families, mutation analyses were also performed in the absence of MSI/abnormal IHC.

Molecular methods

MMR mutation analyses included heteroduplex identiﬁcation followed by DNA sequencing of the actual MMR gene(s).

Analysis of gross deletions and duplications was performed by multiplex ligation-dependent probe ampliﬁcation assay (MLPA;

SALSA P003MLH1/MSH2, P008MSH6/PMS2and P072-MSH6;

MRC-Holland, http://www.mrc-holland.com). Results for PMS2 exon 13e15 probes were disregarded because many related sequences are present in the genome and the probes provided very variable results. Sequencing analyses were performed on an ABI Genetic Analyzer model 3100 or 3130 (Applied Biosystems, Carlsbad, CA, USA), and DNA sequences were computed using SeqScape v2.5 software (Applied Biosys- tems). Primer and sequence details are available on request. In some cases of putative splice effects, cDNA analyses ofMLH1, MSH2, MSH6andPMS2were performed. The molecular analyses were performed according to standard procedures and manufacturers’instructions. Methods used varied over time and between the different laboratories involved. It was beyond our means to reanalyse the whole series so that one method was applied to all cases for the present report.

Classification of DNA variants

Reference sequences used were as follows (GeneBank http://

www.ncbi.nlm.nih.gov/genbank):MLH1, NT_022517 (transcript:

NM_000249.2);MSH2, NT_022184 (transcript: NM_000251.1);

MSH6, NT_0221844 (transcript: NM_000179.1); PMS2, AC005995.3 (transcript: BC093921.1). Detected DNA variants were checked against published mutations in the following websites: http://www.insight-group.org (LOVD: Leiden Open Variation Database), https://portal.biobase-international.com/

hgmd/pro/start.php (Human Gene Mutation Database), Pub Med and http://www.med.mun.ca/MMRvariants.¹⁷ Mutations causing direct stop/nonsense, frameshifts, splice defects and large insertions/deletions were considered deleterious. Missense mutations or small in-frame deletions were subjected to segregation analysis when possible.¹⁸If a review of the international databases or segregation analyses strongly suggested the variant to be deleterious, the mutations were scored accordingly. The reasons for scoring of each mutation are given in table 1. All other DNA variants were considered part of normal variation or the information available on the variant and family was insuf- ﬁcient for conclusive scoring. These variants were excluded from the report.

Clinical classification

All families in which an MMR mutation (ie, with confirmed Lynch syndrome) had been detected were reclassified according to clinical criteria with the information obtained as of November 2009. Thus the classification does not reflect the starting point with the information at hand at referral, but rather the information obtained after having expanded all Lynch syndrome families and verified all relevant diagnoses for all family members in the medical files or cancer registry. The families were classified according to the AMSI, AMSII or BII criteria. Furthermore, the scoring for BII criteria was specified according to the subgroups given in Umar et al⁶: BII_1 (CRC<50 years), BII_2 (synchronic/metachronic cancers), BII_4 (two affected relatives, one <50 years) and/or BII_5 (relatives with HNPCC-associated tumours). For precise definitions of groups as applied, see Umar et al.⁶ BII_3 includes MSI, which was a selection criterion for DNA analysis, and was not used to categorise mutation-carrying kindreds revealed this way. Also, MSI is a laboratory finding and not a clinical criterion. The combined BII criteria were possibly too close to our inclusion criteria for the total cohort studied, and scoring for the combined BII criteria could not be considered a result.

RESULTS

Sixty-nine different mutations were identiﬁed in a total of 129 families. Of these, 31 (45%) were detected inMSH2, 19 (27%) in MSH6, 15 (22%) inMLH1, and four (6%) inPMS2. Sixty-ﬁve (50%) of the families had a mutation in MSH2, 33 (26%) in MSH6, 23 (18%) inMLH1, and eight (6%) inPMS2. The total numbers of mutation carriers were 514, of whom 248 (48%), 146 (28%), 98 (19%) and 22 (4%) had a mutation inMSH2,MSH6, MLH1andPMS2, respectively.

Frameshift mutations (n¼24) and splice defects (n¼18) were the most common aberrations. Other types of mutations were nonsense mutations creating new stop codons (n¼13), large genomic (exon) deletions (n¼8), in-frame deletions of three nucleotides (n¼3) and missense mutations (n¼3). There were no indications that the nature of mutations differed between the different genes, and no further statistical analyses based on the nature of the mutation were undertaken.

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Table 1 Deleterious mismatch repair (MMR) mutations in the Norwegian population^{22 31} Type of

mutation/ gene Mutation

Effect of mutation

(verified or predicted) Family No

No of mut+

IHC (missing protein)

Inclusion criteria:

Amsterd.zz Inclusion

criteria: BIIxx Ref.

Frameshift

MLH1e1 c.39_40dupGA p.Thr14ArgfsX3 H1855 (D4354) 6 MLH1/PMS2 I & II 1, 2, 4 & 5 LOVD

T343 1 MLH1/PMS2 I & II 1, 2, 4 & 5

D20 1 MLH1/PMS2 I & II 1, 2, 4 & 5

MLH1e5 c.413delC p.Pro138LeufsX21 U82517 1 MLH1/PMS2 I & II 1, 2, 4 & 5 {{

MLH1e10 c.866_867delAC p.His289ProfsX16 H836 2 MLH1/PMS2 I & II 1, 2, 4 & 5 LOVD MLH1e13 c.1411_1414delAAGA p.Lys471AspfsX19 H892* 3 MLH1/PMS2&

MSH2/MSH6

II 1& 5 LOVD

MLH1e16 c.1771dupG p.Asp591GlyfsX1 U97760 3 MLH1&PMS2 I & II 1, 2, 4 & 5 {{

MSH2e2 c.229_230delAG p.Ser77CysfsX3 H3323 1 MSH2/MSH6 0 1 LOVD

MSH2e4 c.675_678delAGAA p.Thr225ThrfsX19 D2679 1 MSH2/MSH6 II 1, 4 & 5 {{

MSH2e6 c.969_970delTC p. Gln324ValfsX8 U85816 2 MSH2/MSH6 I & II 1, 2, 4 & 5 {{

D2033 1 MSH2/MSH6 I & II 1, 2, 4 & 5

MSH2e7 c.1204delC p.Gln402LysfsX10 H677 1 MSH2/MSH6 II 1, 4 & 5 LOVD

MSH2e10 c.1594dupG p.Val532GlyfsX3 D139 5 MSH2/MSH6 I & II 1, 2, 4 & 5 LOVD MSH2e11 c.1705_1706delGA p.Glu569IlefsX1 D2938 4 MSH2/MSH6 II 1, 2, 4 & 5 LOVD MSH2e13 c.2120_2122delGCA

insCGGGCTAAGAAGTG

p.Cys707SerfsX2 D1570 5 MSH2/MSH6 I & II 1, 2, 4 & 5 {{

MSH6e4 c.900dupG p.Lys301GlufsX11 U88612 2 MSH2/MSH6 II 2, 4 & 5 {{

MSH6e4 c.1405delT p.Tyr469IlefsX11 S254 9 normaly II 5 {{

MSH6e4 c.1943delG p.Ser648MetfsX5 H2327 3 MSH2/MSH6 0 1, 2 & 4 {{

MSH6e4 c.2604delG p.Met868IlefsX5 D1731 3 MSH6 II 2 & 5 {{

MSH6e5 c.3195_3199delCTATA p.Asn1065LysfsX4 D2115 5 MSH6 II 1, 4 & 5 LOVD

MSH6e5 c.3261dupC p.Phe1088LeufsX5 H1408 1 MSH6 I & II 1, 4 & 5 LOVD

S631 7 MSH6 0 1 & 2

S1108 4 MSH6 II 1, 2 & 4

T02 2 MSH2/MSH6 I & II 1, 2, 4 & 5

MSH6e5 3261delC p.Phe1088ProfsX2 D867 8 MSH6 0 2 & 5 LOVD

MSH6e6 c.3514dupA p.Arg1172LysfsX4 U94618 1 MSH6 II 2 LOVD

MSH6e9 c.3804dupA p.Cys1269MetfsX5 U61010 7 MSH2/MSH6 0 2 & 5 LOVD

U98731 2 MSH6 II 2, 4 & 5

U1000922 2 MSH6 0 0

U1003522 1 MSH6 0 1 & 5

D1151 4 MSH6 0 2 & 5

S889 1 MSH6 0 2

MSH6e9 c.3832_3845del14 p.Pro1278_1282delfsX6 U1000116 1 MSH6 II 4 & 5 {{

PMS2e7 c.736_741delCCCCCT insTGTGTGTGAAG

p.Pro246CysfsX2 U97751 1 PMS2 0 1 & 5 LOVD

PMS2e14 c.2382dupT p.Gly795TrpfsX29 T92 3 PMS2 0 2 & 5 {{

Splice defect

MLH1int9 c.790+1G/A Skipping of exon 9-10 H285 2 ND 0 1, 4 & 5 LOVD

MLH1int9 c.791e2A/G Splice defect T04 (S639 & H1547)) 8 MLH1/PMS2 I & II 1, 2, 4 & 5 LOVD MLH1e10 c.793C/T p.Arg265Cysz D490 10 MLH1/PMS2 I & II 1, 2, 4 & 5 LOVD MLH1e15 c.1731G/C Skipping of exon 15x U1001245 3 MLH1/PMS2 I 1, 2, 4 & 5 LOVD

(c.1731G/A) MLH1int15 c.1731+1G/C Splice defect D1532 1 MLH1/PMS2 II 1, 2, 4 & 5 LOVD MSH2e5 c.815C/T r.(¼)+(¼; 793_942del){ S403 5 MSH2/MSH6 0 2, 4 & 5 LOVD

MSH2int5 c.942+3A/T r.(¼)+(793_942del){ H07 5 ND II 1, 2, 4 & 5 LOVD

H892* 3 MSH2/MSH6 II 1, 2, 4 & 5

H1503 (S551) 4 MSH2/MSH6 0 1, 2, 4 & 5

H1598 (S583) 3 MSH2/MSH6 II 1, 2, 4 & 5

H2215 4 MSH2/MSH6 I & II 1, 2, 4 & 5

H2280 1 MSH2/MSH6 0 1, 2, 4 & 5

U101185 1 MSH2/MSH6 II 1, 4 & 5

T059 3 MSH2/MSH6 0 1, 4 & 5

T073 9 MSH2/MSH6 0 1, 2, 4 & 5

D637 3 MSH2/MSH6 0 4 & 5

D1211 11 MSH2/MSH6 I & II 1, 2, 4 & 5

D4522 2 MSH2/MSH6 0 1, 2 & 5

MSH2int6 c.1076+1G/A Skipping of exon 6 H1903 5 MSH2/MSH6 0 1, 4 & 5 LOVD

D1773 5 MSH2/MSH6 0 2 & 5

MSH2int7 c.1277e2A/G r.(¼, 1277_1386del){ S577 9 MSH2/MSH6 I & II 1, 2 & 4 LOVD Continued

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Table 1 Continued Type of

Effect of mutation

No of mut+

Inclusion criteria:

S612 4 MSH2/MSH6 0 1, 2 & 4

D671 2 MSH2/MSH6 II 1, 2, 4 & 5

MSH2int10 c.1661+1G/A Splice defect D470 2 MSH2/MSH6 II 1, 2, 4 & 5 LOVD MSH2e11 c.1759G/C r.(¼, 1662_1759del)x{ S959 2 MSH2/MSH6 0 1, 2 & 4 LOVD MSH2int11 c.1759+2T/A Deletion exon 12, 13 D971 1 MSH2/MSH6 I & II 1, 2, 4 & 5 LOVD MSH2e12 c.1979A/G r.(¼, 1979_2005del){ U74987 1 MSH2/MSH6 0 1, 2, 4 & 5 31 MSH2int12 c.2006e1G/C splice defect D2013 3 MSH2/MSH6 I & II 1, 2, 4 & 5 LOVD MSH2int15 c.2634+1G/T r.(¼, 2459_2634del) H246/275 10 ND I & II 1, 4 & 5 LOVD MSH6int7 c.3647e2A/C r.(¼, 3646_3647ins3646

+1_3646+492){ S819

(U100998&U104021)

10 MSH6 II 1, 2 & 4 LOVD

D686 7 MSH6 II 0

PMS2intr5 c.537+1G/T Splice defect H3118 2 PMS2 0 1 {{

PMS2int9 c.989e1G/T r.(¼)+(989_1144del, 989_1015del){

S90 4 normal (MSI)** 0 1, 2 & 4 22

S335 1 normal (MSI)** II 1, 2

S350 4 PMS2 I & II 1 & 4

S1147 2 normal (MSI)** 0 1

D3786 5 PMS2 I & II 1, 2, 4 & 5

Stop codon

MLH1e2 c.184C/T p.Gln62X H321 8 ND I & II 1, 4 & 5 LOVD

H480 4 ND I & II 1, 2, 4 & 5

H487 3 MLH1/PMS2 I & II 1, 2, 4 & 5

D498 9 MLH1/PMS2 I & II 1, 2, 4 & 5

D874 4 MLH1/PMS2 I & II 1, 2, 4 & 5

D1704 5 MLH1/PMS2 I & II 1, 2, 4 & 5

MSH2e1 c.142G/T p.Glu48X U1101385 1 MSH2/MSH6 0 1 & 5 LOVD

U101386 1 MSH2/MSH6 II 1, 4 & 5

MSH2e1 c.181C/T p.Gln61X D3959 3 MSH2/MSH6 II 1 & 5 LOVD

MSH2e2 c.226C/T p.Gln76X D271 2 MSH2/MSH6 0 1, 2, 4 & 5 LOVD

MSH2e12 c.1857T/G p.Tyr619X D3648 4 MSH2/MSH6 0 1, 2, 4 & 5 LOVD

MSH2e13 c.2038C/T p.Arg680X U59124 1 MSH2/MSH6 I 1, 2, 4 & 5 LOVD

D414 7 MSH2/MSH6 I & II 1, 2, 4 & 5

MSH2e14 c.2275G/T p.Gly759X D1661 4 MSH2/MSH6 0 1, 2, 4 & 5 LOVD

MSH6e3 c.467C/G p.Ser156X D1651 1 MSH6 0 5 LOVD

MSH6e4 c.718C/T p.Arg240X D4216 2 MSH6 0 1, 2, 4 & 5 LOVD

MSH6e4 c.1444C/T p.Arg482X S407 5 MSH2/MSH6 I & II 1, 2 & 4 LOVD

S1003 10 MSH6 0 5

MSH6e4 c.1483C/T p.Arg495X S363 5 MSH2/MSH6 II 2 & 4 LOVD

MSH6e4 c.2731C/T p.Arg911X D1316 7 MSH6 0 1, 2 & 5 LOVD

MSH6e9 c.3991C/T p.Arg1331X H1522 5 ND 0 1, 2 & 4 LOVD

D1826 10 MSH6 0 5

Exon deletion

MLH1 c.546-?_790+?del del exon 7e9 S499 (H1102) 11 MLH1/PMS2 I & II 1, 2, 4 & 5 LOVD

D2020 4 MLH1/PMS2 I & II 1, 2, 4 & 5

MLH1 c.1732-?_1896+?del del exon 16 H2094 1 ND I 1, 4 & 5 LOVD

MSH2 c.1-?_366+?del del exon 1e2 S541 2 MSH2/MSH6 0 1, 2 & 4 LOVD

MSH2 c.1-?_1076+?del del exon 1e6 H592 3 MSH2/MSH6 0 1, 2, 4 & 5 LOVD

D1718 4 MSH2/MSH6 0 1, 2, 4 & 5

MSH2 c.1-?_1276+?del del exon 1e7 U81431 2 MSH2/MSH6 I & II 1, 2, 4 & 5 LOVD

MSH2 c.1-?_1661+?del del exon 1-10 D3824 3 MSH2/MSH6 0 1 & 5 LOVD

MSH2 c.212-?_1276+?del del exon 2-7 H346 3 ND I & II 1, 2, 4 & 5 LOVD

H496 1 MSH2/MSH6 II 1, 4 & 5

H1110 (S604) 9 MSH2/MSH6 II 1, 2, 4 & 5

S81 5 MSH2/MSH6 I & II 1, 4 & 5

S281(U1002732) 8 MSH2/MSH6 I & II 1, 2, 4 & 5

S649 6 MSH2/MSH6 II 1, 2 & 4

D2107 7 MSH2/MSH6 I & II 1, 2, 4 & 5

MSH2 c.367-?_645+?del del exon 3 H400 4 MSH2/MSH6 I & II 1, 4 & 5 LOVD

In-frame deletion

MSH2e3 c.571_573delCTC p.Leu191del H1294 1 MSH2/MSH6 I & II 1, 2, 4 & 5 {{

H2544 1 MSH2/MSH6 0 1 & 5

H3517 1 ND 0 4

Continued

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Details on the prevalence of each mutation are given in table 1. Forty-nine of the 69 mutations were exclusively found in one family. There were no mutations that were frequent enough to have a signiﬁcant effect on the distributions observed between the different genes. The most recurrent mutation, MSH2 c.942+3A/T, was found in 12 apparently unrelated families (49 people) from different geographical locations and has been described in other populations.^19e21 It has possibly been introduced more than once into our population. The majority of the remaining recurrent mutations could be traced to conﬁned geographical areas and were considered to be branches from a common origin (founder mutations). Families with deleterious PMS2 mutations were limited. One single mutation (c.989e1G/T)²²accounted for the majority ofPMS2 mutation-carrying kindreds, all from the same (small) area.

Average numbers of demonstrated mutation carriers per family were similar for all mutations; details are shown in table 2.

Tumour tissue was available for IHC and MSI analysis for most of the families later demonstrated to have an MMR mutation. The IHC results are shown in table 1. All but ﬁve families showed abnormal IHC corresponding to the gene mutated. Tumours from three ofﬁve kindreds with the founder PMS2splice variant, c.989e1G/T, mentioned above expressed PMS2normally but showed MSI (MSI-high). Similarly, tumour

tissue from one family with a missense mutation in MLH1 (c.245C/T (p.Thr82Ile)) showed normal IHC and MSI (MSI- high). Apparently normal expression ofMLH1indicated by IHC in MSI-high tumours is in agreement with another report.²³In the family with the c.1405delT, inMSH6, the tumour showed normal IHC and was microsatellite stable (MSS).

The mutation-positive families that fulﬁlled the various clinical criteria when reclassiﬁed are detailed in table 2. Thirty-eight per cent ofMSH2families, 12% ofMSH6families, 78% ofMLH1 families and 25% of PMS2 families met the AMSI criteria.

Corresponding sensitivity for the AMSII criteria for identifying mutations in the different genes were 62%, 48%, 87% and 38%.

Similarly, each of the clinical Bethesda criteria had low sensitivity for identifyingMSH6andPMS2mutations.

DISCUSSION

In this study in which all national activity was compiled, we found that most families withMLH1mutations were identified by any of the clinical criteria used. The criteria that included extracolonic cancers (AMSII) identified two out of threeMSH2 mutations, whereasMSH6mutations were not identified with reasonable sensitivity by any of the single clinical criteria. As these results were obtained after expanding all mutation- Table 1 Continued

Type of

Effect of mutation

No of mut+

Inclusion criteria:

U90087 1 MSH2/MSH6 II 1, 2, 4 & 5

U1000173 2 MSH2/MSH6 II 1, 4 & 5

T382 3 MSH2/MSH6 0 1, 4 & 5

MSH2e12 c.1786_1788delAAT p.Asn596del D554 19 MSH2/MSH6 I & II 1, 2, 4 & 5 LOVD

D853 5 MSH2/MSH6 I & II 1, 2, 4 & 5

D3618 5 MSH2/MSH6 I & II 1, 2, 4 & 5

D3667 3 MSH2/MSH6 I & II 1, 2, 4 & 5

D3707 7 MSH2/MSH6 I & II 1, 2, 4 & 5

D4202 2 MSH2/MSH6 II 1, 2, 4 & 5

MSH6e4 c.2302_2304delCCT p.Pro768del H801 2 MSH2/MSH6 0 1, 2, 4 & 5 LOVD

4/14 H2160 5 ND 0 2, 4 & 5

S149 5 MSH2/MSH6 0 1 & 5

S647 3 MSH2/MSH6 II 2

Missense

MLH1e3 c.245C/T p.Thr82Ile S420yy 6 normal (MSI)** II 1, 4 & 5 LOVD

MLH1e16 c.1823C/A p.Ala608Asp S581yy 2 MLH1/PMS2 II 1, 2 & 4 LOVD

MSH6e4 c.2906A/G p.Tyr969Cys D2955yy 6 MSH6 I & II 1, 2, 4 & 5 LOVD

*Two pathogenic mutations in two branches in the same family.

yNo indications from IHC or microsatellite instability.

zReported to affect splicing and stability.

xLast nucleotide in exon; reported to cause skipping of exon.

{Shown in present study to give aberrant splicing.

**Normal protein expression, but microsatellite instability.

yyCosegregation with disease.

zzAmsterdam I and/or Amsterdam II.

xxBethesda II (revised), see text for details.

{{Not found to be reported in databases.

IHC, immunohistochemical analysis; LOVD, Leiden Open Variation Database (http://www.insight-group.org/mutations/); mut+, mutation carriers; ND, not done.

Table 2 Summary of deleterious variants according to gene, number of mutation carriers and which clinical criteria are fulfilled

Gene No of families No of mut+ Fraction of mutations (in %) No of mut+/family AMSI AMSII BII_1 BII_2 BII_4 BII_5

MSH2 65 248 50 3.82 25 (10.38) 40 (10.62) 61 (10.94) 46 (10.71) 58 (10.89) 57 (10.88)

MSH6 33 146 26 4.42 4 (10.12) 16 (10.48) 15 (10.45) 24 (10.72) 16 (10.48) 22 (10.67)

MLH1 23 98 18 4.26 18 (10.78) 20 (10.87) 23 (11.00) 20 (10.87) 23 (11.00) 23 (11.00)

PMS2 8 22 6 2.75 2 (10.25) 3 (10.38) 7 (10.88) 4 (10.50) 3 (10.38) 3 (10.38)

Total 129 514 100 3.98

AMSI/II, Amsterdam I/II criteria; BII, Bethesda II criteria; mut+, mutation carriers.

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carrying kindreds, we consider them to be maximum estimates.

The sensitivities for detecting mutation-positive families upon referral were lower.

The most sensitive single clinical criterion for identifying MSH6mutation carriers was the presence of two independent primary cancers (BII_2) (table 2). This information is, however, awaiting detailed validation of diagnoses in the families and may not be easily obtainable when interviewing a family member.

We have recently reported that, when applied to a consecutive series of unselected patients with CRC, the sensitivities of AMSII and BII criteria were as low as 25% and 50%, respectively.¹²Moreover, awareness of hereditary cancer among clinicians involved in diagnosis and treatment of CRC is low, and families actually meeting the criteria may not be identified.²⁴ These points highlight the challenges associated with using family history for detecting families with MMR mutations. Our combinedfindings support the suggestion by the Mallorca group to apply IHC and/or MSI analysis to all CRCs to identify MMR mutation carriers.²⁵ As MSH6 mutation carriers are likely to develop extracolonic cancers, it may be justified to suggest that all cancer phenotypes associated with Lynch syndrome should be subjected to IHC and/or MSI analysis and subsequent DNA mutation analysis. Until such studies have been performed, we remain cautious when discussing the prevalence of MSH6 mutations. Correspondingly, the current estimates of penetrance/expression ofMSH6 mutations may be (partly) derived from families fulfilling current clinical criteria.^{26 27}These estimates may be misleading, as they may reflect the criteria used to select the families from which the estimates were derived.

MLH1 mutations were less common than assumed from previous reports,⁸ and MSH2 mutations accounted for almost half of all kindreds with a mutation. Despite the fact that the criteria used were insensitive for detectingMSH6mutations, the number ofMSH6mutation-carrying kindreds were higher than MLH1mutation-carrying kindreds.

None of the mutations were common enough to affect the distribution signiﬁcantly, with respect to neither number of mutation-carrying kindreds nor number of mutation-carrying people. Despite the difference in prevalence of mutations in the different genes, the mean number of mutation carriers per family was similar for all the genes.

A Danish study reported a relatively high prevalence ofMSH6 mutations.²⁰ If this were due to similarities between these neighbouring populations, we would have expected to detect founder mutation(s) in both populations, but this was not the case. The reason for the similar results may be the study designs.

Both studies applied wide criteria for IHC and MSI analysis.

The number of carriers ofPMS2mutations was insufﬁcient for sophisticated statistical analysis. Part of the explanation may be that testing forPMS2mutations has not been available for as long as testing for mutations in the other genes. Also, most of the few PMS2 mutation carriers were included in several branches of one old family. Some of these branches were not identiﬁed by IHC, but the tumours displayed MSI (MSI-high).

Thus, by performing only IHC and not MSI analysis to prescreen for mutation testing, a few mutations may have been missed. This indicates that MSI analysis is of importance if IHC shows normal expression of all MMR genes. MMR mutations cannot be excluded if neither analysis has been performed.

Technical problems involved in DNAPMS2 mutation analyses are well known.^28e30 Current procedures (including both technical aspects and clinical criteria) may be insufﬁcient to detect PMS2mutations, and current estimates of prevalence ofPMS2 mutations may be too low.

IHC was used as a selection criterion for mutation analysis and could not be scored as a result. The result of all efforts to examine selected families without abnormal IHC or MSI for mutations was the identiﬁcation of a single mutation-carrying family (family S254, MSH6 c.1405delT). The study was not designed to assess sensitivity of IHC/MSI, and we will not discuss this further. Our impression from other reports is, however, that IHC and MSI analyses are more sensitive than any clinical criteria for identifying kindreds carryingMSH2 or MSH6mutations, in particular,^{11 12}and the present report is in keeping with that notion.

In conclusion, we observed that 87% of families with an MLH1mutation, 62% with anMSH2 mutation, but less than half of families with anMSH6orPMS2mutation were identified by the AMSII criteria. Each of the clinical Bethesda criteria when considered individually also showed low sensitivity. We have, however, previously demonstrated that these criteria were neither sensitive nor specific in an unselected series of CRC cases. Our combined observations indicate that the prevalence of MSH6 mutations may be higher than currently assumed, and their penetrance and expression may differ from what is currently assumed. These findings are in keeping with the Mallorca guidelines, which recommend that MSI analysis and/or IHC should be performed on all CRCs.²⁵In addition, we suggest that such testing should be applied to all incident cancers in the Lynch syndrome tumour spectrum to increase the rate of detection of MMR mutation carriers.

AcknowledgementsWe thank the families that have actively contributed to this study, by giving us information and permission to study them. The assistance of the laboratory staff and clinicians in all Norwegian medical, genetic and pathological laboratories involved in testing for Lynch syndrome (HNPCC) is greatly appreciated.

Competing interestsNone.

ContributorsConception: WS, PM. Clinical data: EMG, PM, AS, LFE, IB, CJ, LM.

Mutation analyses including MSI: WS, BIH, PAA, SA, LASL, BG. IHC: HA, IMB-L.

Manuscript writing: WS, PM, EMG. Approval of final manuscript: all.

Provenance and peer reviewNot commissioned; externally peer reviewed.

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doi: 10.1136/jmg.2010.077677

2010

2010 47: 579-585 originally published online June 28,

J Med Genet

Wenche Sjursen, Bjørn Ivar Haukanes, Eli Marie Grindedal, et al.

mutation carriers

MSH6 are not sensitive enough to identify